Biofilm Signaling, Composition and Regulation in Burkholderia pseudomallei

The incidence of melioidosis cases caused by the gram-negative pathogen Burkholderia pseudomallei (BP) is seeing an increasing trend that has spread beyond its previously known endemic regions. Biofilms produced by BP have been associated with antimicrobial therapy limitation and relapse melioidosis, thus making it urgently necessary to understand the mechanisms of biofilm formation and their role in BP biology. Microbial cells aggregate and enclose within a self-produced matrix of extracellular polymeric substances (EPSs) to form biofilm. The transition mechanism of bacterial cells from planktonic state to initiate biofilm formation, which involves the formation of surface attachment microcolonies and the maturation of the biofilm matrix, is a dynamic and complex process. Despite the emerging findings on the biofilm formation process, systemic knowledge on the molecular mechanisms of biofilm formation in BP remains fractured. This review provides insights into the signaling systems, matrix composition, and the biosynthesis regulation of EPSs (exopolysaccharide, eDNA and proteins) that facilitate the formation of biofilms in order to present an overview of our current knowledge and the questions that remain regarding BP biofilms.


Cyclic-di-GMP Signaling
C-di-GMP is a bacterial universal intracellular secondary signaling molecule [36][37][38]. In bacterial biofilm formation, c-di-GMP is known to regulate genes responsible for synthesizing EPS components; extracellular polymeric exoenzymes, polysaccharides, and adhesins [39,40]. In addition, c-di-GMP enhances bacterial adhesion and represses bacterial motility, further promoting biofilm production [32,33,41,42]. Furthermore, depletion of c-di-GMP levels has been reported to trigger the dispersal of biofilms. For instance, inhibition of the final step of the denitrification pathway has been implicated in inducing biofilm dispersal [43]. Nitrate levels have been reported to significantly affect biofilm formation in BP, as they ultimately determine the fate of c-di-GMP [33]. The denitrification process, which involves the reduction of nitrate to nitrogen, is important in BP biofilms as it provides an alternative energy source under oxygen-limited conditions [33,44]. The impact of inhibiting the denitrification pathway on biofilms was recently evaluated in B. thailandensis, a species closely related to BP [43]. Inhibiting the final step of denitrifying nitrous oxide to nitrogen catalyzed by nitrous oxide reductase leads to the accumulation of nitrous oxide, which in turn reduces c-di-GMP levels that ultimately trigger the dispersal process [43,45]. As for BP, a recent transcriptome analysis between high and low BP biofilm-forming isolates revealed the overexpression of nitrous oxide reductase, bpsl1607, in the high biofilm-forming isolate. Furthermore, studies on BP 1026b isolate mutants involving a two-component, nitrate-sensing system in the form of narX-narL (equivalent to bpsl2313-bpsl2314) have further confirmed the regulation of the denitrification pathway in c-di-GMP production and biofilm formation [44].
The synthesis and breakdown of c-di-GMP in most bacteria are regulated by diguanylate cyclase (DGC) and phosphodiesterase (PDE), respectively. The activity of both proteins is affected by environmental cues, in agreement with the transition of bacteria from planktonic to biofilm state being regulated by c-di-GMP in response to changes in environmental stimuli [46][47][48]. DGC contains the conserved GGDEF domain, while PDE

Fig. 1. Schematic diagram representing four stages of biofilm formation (1) surface bacterial attachment, (2) microcolony formation, (3) maturation of biofilm architecture, and (4) dispersion of cells into the planktonic state (adapted from [15-17]).
contains a conserved EAL or HD-GYP domain [11,49]. DGC catalyzes the synthesis of c-di-GMP from the condensation of two GTP molecules, while PDE catalyzes the hydrolysis of c-di-GMP, resulting in two GMP molecules [50,51]. Burkholderia cenocepacia is another Burkholderiaceae family member and closely related species to BP, and a number of genes encoding proteins that are homologous across the Burkholderia group responsible for the synthesis of c-di-GMP in B. cenocepacia have been identified and tabulated (Table 1) [51].
In BP, a putative DGC (bpss2342 or Bp1026b_II2523) that contains a conserved GGDEF domain was reported to influence the biofilm formation in a temperature-dependent manner in which increased biofilm formation was observed among mutant colonies grown at 37 o C compared to 30 o C [41,52]. This observation highlights the correlation between c-di-GMP synthesis and environmental factors regulating BP's biofilm formation. Furthermore, the Bp1026b_II2523 (bpss2342) mutant was shown to affect various biological systems such as polysaccharide biosynthesis and several secretion systems (T3SS-3, T3SS-2, T6SS-3, and T6SS-6) and biosynthetic gene clusters (BGCs) that are involved in non-ribosomal peptide and polyketide synthesis and, predicted to encode small metabolites contributing to biofilm development [52]. Apart from that, the cdpA gene in BP KHW (corresponding to bpsl1263 in BP K96243) encoding phosphodiesterase proteins that contains a conserved EAL has been identified as PDE [42]. The cdpA deletion mutant was shown to exhibit high levels of cdi-GMP which favor biofilm production through increased exopolysaccharide and cellular aggregation [42]. Furthermore, higher expression of the cdpA gene was observed for BP exposed to exogenous sodium nitrate. This led to the upregulation of PDE activity which contributed to reduced c-di-GMP levels and, subsequently, poor biofilm formation [33]. Recently, a c-di-GMP signaling cascade mediated by a pdcABC operon that can regulate virulence, motility, and biofilm formation was reported for B. thailandensis [53]. pdcA encodes a DGC protein that produces c-di-GMP and is regulated by PdcC (phosphate-accepting response regulator). The phosphorylated PdcC inhibits PdcA by binding to its PAS domain. PdcB is a phosphatase that increases the activity of PdcA through dephosphorylation of PdcC [53]. Interestingly, an operon in BP shares high sequence identity with pdcABC ( Table 1), suggesting that BP may share a similar pathway in modulating c-di-GMP levels.
Several genes encoding proteins that contain the conserved GGDEF and EAL domains have been annotated in the BP genome (https://www.burkholderia.com/) and Plumley et al. [41,54]. The proteins BPSL1306, BPSS0136, BPSS1297, BPSS1971, and BPSS2342 were predicted to carry the GGDEF domain with high sequence identity ( Supplementary Fig. S1) while BPSL0358, BPSL0887, BPSL1286, BPSL1635, BPSL2744, and BPSS0799 have the EAL domain ( Supplementary Fig. S2). Meanwhile, five other proteins (BPSL0602, BPSL1080, BPSL1263, BPSS0805, and BPSS2318) contain both the conserved GGDEF/EAL domains (Table 1) [41,54]. By modulating the level of c-di-GMP, the GGDEF and EAL domains were reported to exert control at the transcriptional and post-translation level in regulating the expression of cell surface components (e.g., flagella, adhesins, pili, exopolysaccharides) ( Fig. 2) [41]. Nitrate level and temperature have been proven to affect the DGC gene Bp1026b_II2523 (bpss2342) and cdpA, respectively. Nonetheless, the function of other predicted genes that encode DGC and PDE enzymes containing GGDEF/EAL domains in BP needs to be further characterized in terms of their correlation to other specific environmental cues. It is known that proteins containing GGDEF/EAL domain generally assemble an N-terminal sensory domain which may respond to specific environmental stimuli (oxygen, light, nitric oxide, etc.) in regulating the enzyme activity that may determine the production level of c-di-GMP [55][56][57]. Based on currently available reports, we proposed the c-di-GMP synthesis mechanism and its functional properties during BP biofilm formation and the factors that may influence the biosynthesis of c-di-GMP in enhancing the transition from free-living planktonic cells to sessile cells, as illustrated in Fig. 2.

Quorum Sensing (QS) Signaling
Quorum sensing is also a crucial signaling system involved in forming biofilms. Autoinducers produced by bacteria serve as chemical signal molecules and are released according to cell density [59,60]. QS is utilized by both gram-positive and gram-negative bacteria [60]. In most Burkholderia spp., inhibition of this signaling system negatively affects biofilm formation, making the QS signaling system a suitable target for antimicrobials or antibiofilm agents [61,62].
N-acyl-homoserine lactones (AHLs) are the most common QS signaling molecule utilized by most gram- negative bacteria, including BP [63]. AHL signaling molecules are encoded by a class of genes that are homologous to the luxI and luxR of Vibrio fischeri and have been reported to mediate QS systems [65,66]. luxI encodes AHL synthetases that are required for the synthesis of related signaling molecules, while LuxR family proteins serve as AHL molecular receptors [66,67]. AHL autoinducers interact with the LuxR proteins to regulate the expression of genes that control relevant biological phenotypes, including biofilm formation [66,67]. Similar QS systems in Pseudomonas aeruginosa, namely lasIR and rhlRI, are homologous to the LuxI-LuxR [68,69]. In BP, the BpsI-BpsR QS system was reported as a homolog of LuxI-LuxR [70] and positively regulates biofilm formation. BP owns three QS systems that produce AHL molecules, namely QS-1 (encoded by BpsI-BpsR), QS-2 (BpsI 2 -BpsR 2 ), and QS-3 (BpsI 3 -BpsR 3 ), which produce three types of AHLs, N-octanoylhomoserine lactone (C8HL), N-(3-hydroxy-octanoyl) homoserine lactone (OHC8HL), and N-(3-hydroxy-decanoyl) homoserine lactone [66,71]. C8HL is synthesized by N-acyl-homoserine lactone synthase, encoded by bpss0885 (pmlI), which is also known as BpsI [54,66]. The remaining two AHLs are mainly produced by BpsI 2 (encoded by BPSS1180 in BP K96243) and BpsI 3 (BPSS1570), respectively, which are paralogs to BpsI [54,66]. The expression of the three BpsI enzymes is regulated by their corresponding AHL-dependent transcription regulators BpsR, BpsR 2 , and BpsR 3 , respectively. BP strains lacking the BpsI-BpsR system cannot form biofilm [34,66] while individual bpsR and bpsI mutants have impaired biofilm formation [66,72]. Biofilm formation is restored in the presence of exogenous C8HL. In contrast, the addition of exogenous OHC8HL further suppresses biofilm formation in the mutant strains, indicating that exogenous OHC8HL serves as an antagonist in suppressing the biofilm formed by BP [66,72]. Moreover, it was reported that BpsR 2 is not involved in biofilm formation while BpsR 3 plays a partial role. Unlike BpsR, exogenous OHC8HL was not able to resume full biofilm formation of BpsR 3 mutant. Taken together, only dedicated QS signaling systems (QS-1 and QS-3) in BP were shown to be involved in biofilm formation, suggesting the specificity of AHL-signaling molecules in regulating the biofilm formation mechanism.
Apart from the AHL molecules, BP is known for producing another type of QS molecule known as 4-hydroxy-3-methyl-2-alkylquinolines (HMAQs), which are similar to the Pseudomonas quinolone signal (PQS), 4hydroxy-2-alkylquinolines (HAQs) that are found in P. aeruginosa [73]. The PQS molecule is synthesized by the pqsABCDE operon (pa0996-pa1000) which is homologous to the hhqABCDE (bpss0481-bpss0485) genes in BP [73][74][75]. In P. aeruginosa, anthranilic acid is the precursor molecule for the synthesis of HAQs and is supplied by three different pathways that includes anthranilate synthase encoded by phnAB and trpEG and the degradation of tryptophan through the kynurenine pathway [75]. Similarly, BP produces anthranilic acid via the TrpEG and kynurenine pathway [75,76]. In addition, inhibition of the kynurenine pathway was reported to increase the production of biofilm and reduce motility in BP [76], suggesting the involvement of HMAQs in biofilm formation and as a virulence factor of the bacterium.
In 2008, another quorum-sensing signal, cis-2-dodecenoic acid, also known as Burkholderia diffusible signal factor (BDSF), was reported in B. cenocepacia [77]. The BDSF QS system was reported to exert control towards AHL signaling and biofilm formation and affects the virulency of B. cenocepacia [78][79][80][81]. A rpfF gene that encodes RpfF BC enzyme was found to be responsible for the synthesis of the BDSF, and the production of BDSF is regulated by the RqpSR two-component system [77,82]. A neighboring gene of rpfF, namely rpfR, is a gene encoding protein containing a PAS-GGDEF-EAL domain associated with c-di-GMP synthesis. The deletion of rpfR resulted in increased intracellular c-di-GMP [80]. A further study shows that RpfR is a QS signal receptor that can interact with BDSF and a c-di-GMP phosphodiesterase that interacts with RpfF to inhibit BDSF production [83]. Moreover, RpfR can also act as a c-di-GMP sensor by interacting with the global regulator GtrR [83]. Interestingly, while homologs of RpfR, RqpSR two-component systems and GtrR were identified in BP, no RpfF BC homologs could be detected [84]. However, there have yet to be any reports on RpfR, RqpSR two-component systems, and GtrR in BP. Therefore, it is unknown if the BDSF QS system that regulates c-di-GMP signaling exists in BP. Hence, further studies are warranted for a better understanding of the BDSF QS system and c-di-GMP in regulating the biofilm formation of BP.

Regulation by Small RNAs (sRNAs)
sRNAs modulate protein expression by altering mRNA translation rates or via mRNA degradation [85]. Common metabolic processes regulated by sRNAs include QS, carbon metabolism, and iron homeostasis [86]. These metabolic processes were observed in a recent study on B. cenocepacia J3215 biofilm [85]. In addition, functional characterization of B. cenocepacia J3215 sRNAs through comparison between sRNA mutant and wildtype strains revealed high growth, cellular aggregation, and metabolic activity (upregulation of the tryptophan and phenylacetic acid degradation pathways) among the mutant strains [87]. A recent whole genome-level transcriptome study on B. cenocepacia J2315 biofilm and planktonic states highlighted the abundance of sRNAs in the biofilm transcriptome compared to bacteria in the planktonic state [85], thus suggesting that sRNAs may play a crucial role in the development of a successful biofilm. Fifteen of the identified sRNAs were highly conserved across Burkholderia spp. [85]. Nonetheless, to date, no biofilm-associated sRNAs have been described for BP. Therefore, further investigation to identify the presence and involvement of sRNAs is required to reach a better understanding of biofilm formation.

Biofilm Composition in BP
The EPS matrix forms a natural protection shield for many bacteria, where it enables the bacteria that have changed from the planktonic stage growth mode to live in biofilm in response to various environmental cues and stresses. The formation and degradation of the EPS matrix in the biofilm life cycle are highly regulated and specific J. Microbiol. Biotechnol. mechanisms are involved in the synthesis and degeneration of each of the EPS matrix components. Several major EPS matrix components in BP, including exopolysaccharides, eDNA, and proteins, have been identified. This section provides an overview of the three major EPS components of BP.
Recently, an exopolysaccharide gene cluster of 18 genes (becA-R) was identified. The becA-R is highly conserved within the Burkholderia spp. (B. pseudomallei, B. thailandensis, and B. mallei) [96]. The becA-R cluster encodes various enzymes such as glycosyl transferase, glycosyl hydrolase, capsular polysaccharide UDP-glucose lipid carrier transferase, and mannose-1-phosphate guanylyl transferase, which are required to synthesize exopolysaccharide components within the matrix [97,98]. A transcriptome-level analysis of low and high BP biofilm producers revealed several genes within the becA-R gene cluster (bpsl0603, bpsl0605, bpsl0618, bpsl0619, and bpsl0620) were highly expressed in the high biofilm-producing strain [45]. Apart from the becA-R gene cluster, the wbiA gene cluster that consists of bpsl2670 and bpsl2671 was also identified to be involved in lipopolysaccharide biosynthesis. These genes encode UDP-glucose-4-epimerase and glycosyl transferase family protein, respectively [96]. Nonetheless, the detailed mechanism for exopolysaccharide biosynthesis has yet to be elucidated. Furthermore, several genes within the becA-R cluster encode hypothetical proteins, thus making elucidation of the exopolysaccharide synthesis mechanism more challenging.
Exopolysaccharide production in Burkholderia sp. biofilms is strongly influenced by c-di-GMP and QS signaling molecules [93,97,98]. The transcription regulation factors bpsI, ppk and rpoS were reported to influence the ratio of the monosaccharides glucose, galactose, mannose, and rhamnose of BP biofilm extracted exopolysaccharide [93]. In B. cenocepacia, c-di-GMP regulates exopolysaccharide biosynthesis at the posttranslational level by promoting the binding between the CRP/FNR family transcriptional regulatory protein BCAM1349, (encoded by bcam1349) and the promoter region upstream of the becA-R gene cluster [97][98][99]. Two BP hypothetical proteins (BPSL0616 and BPSL0617) are reported to have a CRP/FNR superfamily domain, with BPSL0617 most likely an ortholog of BCAM1349 (Table 1) [45,96].
Apart from c-di-GMP, N-acyl-homoserine lactone synthase BpsI (AHL synthase or C8HL, BPSS0885), the regulatory protein polyphosphate kinase (PPK, BPSL1366) and an alternative sigma factor S (RpoS, BPSL1505) are known to regulate exopolysaccharide production. Polyphosphate kinase is essential in producing inorganic polyphosphate from ATP which is required in the activation of sugar precursors for exopolysaccharide production [93]. A bpsl1366 mutant showed increased susceptibility to antibiotics due to poor development of the exopolysaccharide framework [93]. AHL synthase and RpoS are crucial in regulating the expression of enzymes involved in the exopolysaccharide biosynthesis pathway to enhance the survival of biofilm cells under adverse conditions [93]. For example, UTP glucose-1-phosphate uridylyltransferase (BPSL2769) and GDP-mannose-4,6dehydratase (WcbK) enzymes are involved in synthesizing UDP-rhamnose. The genes of these proteins are predicted to have a lux box promoter region that responds to BPSS0885 [93]. rpoS regulates a series of enzymes encoded by genes with an RpoS-dependent promoter region, such as glucokinase (BPSL2614), UDP-glucose 4epimerase (BPSL2670), WcbK (BPSL2729), and UDP-glucose-1-phosphate uridylyltransferase (BPSS1682). These enzymes are involved in converting several monosaccharides into galactose and rhamnose [93]. The conversion of glucose into glucose-6-phosphate catalyzed by glucokinase is the first step in biofilm exopolysaccharide synthesis; this highlights the importance of rpoS in regulating exopolysaccharide synthesis in BP biofilms [93]. Furthermore, monosaccharides, particularly rhamnose, contribute to a robust biofilm matrix that significantly limits the diffusion of antibiotics [93]. Therefore, overexpression of bpss0885, bpsl1505, and bpsl1366 accompanied by the accumulation of c-di-GMP, may lead to the formation of a rigid biofilm [93,97]. The mechanisms of c-di-GMP, QS signaling, and RpoS involved in exopolysaccharide biosynthesis for BP biofilm formation are proposed and illustrated in Fig. 3.

Extracellular DNA (eDNA) in EPS
Extracellular DNA (eDNA) is a crucial component of EPS and biofilm development [100][101][102]. eDNA is proposed as a key component of many pathogenic bacteria that form biofilms where it contributes to shielding biofilm against antimicrobial agents, promoting adhesion, and strengthening the integrity of biofilms [101,103,104]. In some bacteria, eDNA is derived from chromosomal DNA that is released from the bacterial cells either by active secretion mediated by QS or through cell lysis [105][106][107]. These mechanisms of eDNA release have been widely described for Staphylococcus epidermidis and P. aeruginosa biofilms [108,109] but are yet to be characterized for BP. However, there is some indication that eDNA production in BP occurs through the extrusion of DNA from living cells, which is controlled by the transcriptional regulator BPSL1887 [110]. Interestingly, recent studies aiming to determine and quantify the components of BP and B. thailandensis biofilms revealed that eDNA and other major components in the biofilms are synthesized by living cells [111]. In addition, strains lacking capsular polysaccharides (CPS I) compensate by producing high levels of eDNA to complete biofilm formation with the abundant eDNA contributing to the thickness of the biofilm matrix [111]. BP bpsl1036 and bpsl1037 mutants that lack the two-component signal transduction systems (TCSTS) implicated in virulence and drug resistance have increased eDNA levels which ultimately promotes biofilm formation in BP [112].
It was reported that eDNA is actively involved during the early stages of biofilm formation, facilitating initial attachment and bacterial aggregation under the planktonic and biofilm states [113,114]. Deoxyribonucleases (DNAses) are able to completely inhibit eDNA activity which is reflected by a reduced biofilm mass. However, inhibition of eDNA activity beyond the initial biofilm formation step shows no significant changes in biofilm mass, due to limited access of DNAse towards eDNA in mature biofilm. Therefore, DNAse treatment could be an appropriate treatment strategy targeting eDNA during the early stages of biofilm infections [113]. The ability of eDNA to defend the biofilm community against antimicrobial agents arises from its chemical properties. The negatively charged eDNA binds to the positively charged ions on antibiotics such as aminoglycosides and antimicrobial peptides, thereby reducing the antimicrobial agents' efficiency in eliminating biofilm-forming pathogens [100,115]. When BP biofilm was subjected to DNase treatment, a drastic reduction in biofilm mass was observed which could not be restored following supplementation with exogenous DNA [113]. A similar observation was noted with Neisseria meningitidis [116], and taken together, implies the importance of BP eDNA in the formation of BP biofilms. More recently, the unraveling of the P. aeruginosa eDNA structure revealed that it was different from P. aeruginosa chromosomal DNA where purine-rich RNAs that were integrated into the eDNA framework enabled crosslinking of the extracellular matrix [117]. Moreover, the formation of G-quadruplexes occurs in eDNA due to the non-canonical Hoogsteen base pairing between thymine/uracil and guanine [118]. The presence of G-quadruplexes in the matrix of P. aeruginosa biofilms was verified by specific antibody binding while the loss of the G-quadruplexes resulted in a lack of eDNA fibers [118]. Hence, this marks the structural specificity of eDNA involved in biofilm formation.
eDNA also exists as a lattice structure stabilized by DNABII proteins [119]. The integration host factor (IHF) and histone-like protein (HU) are two common members of the DNABII protein family that contribute to the lattice structure of the eDNA, thereby increasing the structural stability of the biofilm [120][121][122]. The B. cenocepacia HU and IHF protein orthologs are present in BP [122] (Table 2), indicating similar structural integrity components among the Burkholderia biofilms. Targeting the DNABII proteins via anti-DNABII antibodies

Fig. 3. Proposed BP exopolysaccharide biosynthesis regulation mechanism via c-di-GMP and QS signaling.
Signaling molecules, e.g. c-di-GMP, and QS molecules, e.g., RpoS and AHLs, regulate the development of the EPS components, particularly exopolysaccharides. c-di-GMP is reported to improve the binding between the regulatory protein and the promoter region of the becA-R gene cluster thereby triggering gene expression of the cluster to produce the enzymes that facilitate the synthesis of exopolysaccharides in the EPS.  [123]. These findings highlight the significance of eDNA in the structural integrity of biofilms for most bacteria. Therefore, targeting the eDNA could be a therapeutic strategy to eradicate infections by biofilm-forming pathogens.

Proteins in EPS
The abundance of proteins in EPSs has been examined recently in most bacteria capable of forming biofilms. The function of these proteins to achieve a successful biofilm are diverse [124]. Currently, proteins within EPSs are categorized as enzymes and structural proteins [125]. Numerous enzymes in EPSs are involved in the synthesis or degradation of matrix components. For instance, tyrosine kinase encoded by bceF has been implicated in favoring biofilm formation by mediating the synthesis of exopolysaccharides in B. cepacia IST408 [126]. On the contrary, enzymes that break down the EPS, such as alginate lyase, are involved in the breakdown of exopolysaccharides in P. aeruginosa biofilms [127]. The BP genes encoding tyrosine kinase and alginate lyase annotated in the Burkholderia Genome Database (https://www.burkholderia.com/) are shown in Table 2 [54]. Further investigation is required to assess the enzymatic activity of these enzymes towards the biofilm formed by BP.
EPS proteins that contribute to structural stability include surface-associated proteins, such as pili and flagella, which mediate bacterial initial attachment and adhesion in H. influenzae biofilms and most other bacterial biofilms [124,128]. Pili are known to facilitate bacterial adhesion, motility, DNA transfer, and biofilm formation [129]. BP is known to encode eight types of type IV pili (T4P) [130]. Recently, an uncharacterized type IV piliassociated protein (TFP8) encoded by bpss2185 was reported to be highly expressed during biofilm maturation and dispersal stages, highlighting that bacterial movement is crucial in stabilizing the structure of biofilm in BP [131]. Apart from that, proteomics analysis had discovered an abundance of outer membrane vesicle (OMV) proteins within the matrix of P. aeruginosa and B. multivorans biofilms [102]. The OMV proteins of gram-negative bacteria exist as spherical and bilayer membranes [132]. OMVs released during bacterial growth and and contain lipoproteins, lipopolysaccharides, and outer membrane proteins [133,134]. OMVs are involved in several phenomena such as pathogenesis, bacterial communication, horizontal gene transfer, nutrient capture, bacterialhost interaction, and improvement of coaggregation during biofilm formation [102,134]. Furthermore, OMVs can shield the biofilm community by releasing toxins that can target and affect the host defensive responses [133]. Meanwhile, bpss0093 and bpsl1800, two BP genes, were reported to be highly expressed during biofilm formation [45]. These genes are suggested to encode an outer membrane usher protein, presumably with a similar function to OMVs. Since the abundance of OMVs has been reported within the EPS, utilizing these OMVs to channel the antibacterial agents into the biofilm community serves as a strategy to eradicate biofilm infection [133].

Conclusion and Future Perspective
BP biofilms have been implicated as a virulence factor contributing to the pathogenesis of melioidosis during BP infections. This review systematically presents the genes and proteins that have been shown or predicted to be involved in the biosynthesis of essential B. pseudomallei EPS components. More than 60 genes and proteins representing 1.2% of the total annotated genes of BP have been identified as being involved in its biofilm formation. In this review, we have highlighted several knowledge gaps that require future investigation. These include: (i) the need to elucidate the roles of putative proteins that contain DGC and PDE domains for cyclic-di-GMP signaling; (ii) the determination of specific sRNAs that may have roles in regulating BP biofilm formation; (iii) the characterization of enzymes including hypothetical proteins in the becA-R gene cluster to decode the exopolysaccharide biosynthesis pathway; and (iv) to unravel the mechanistic role of eDNA in biofilm formation and its potential as a target for therapeutics. Furthermore, a systems biology approach could be adopted to characterize further the interrelationship between biofilm formation stages, signaling systems, regulation, and biosynthesis of EPS components.